| OBJECTIVE: To explain genuineness causes of Hedysari Radix in molecular level and construction genetic maps,to establish and optimize the ISSR-PCR reaction system for Hedysari Radix,study on genetic diversity of Hedysari Radix,analysis on correlations between genetic diversity and ecological factors.METHOD:The effects of main elements,i.e. DNA concentration,primer concentration, Mg2+concentration,d NTPs concentration,tempera-ture,Taq polymerase dosage,on ISSR-PCR amplication were tested by central compositedesign and single factor experiment, selected the best annealing temperature determined polymorphic primer and establish and optimize the ISSR-PCR reaction system.Analysis the genetic diversity of Hedysari Radix and genetic diversity parameters by software calculates and UPGMA cluster analysis by MEGA 5.05 software. DNA loci of Hedysari Radix get on cluster analysis and Correlation Analysis between genetic diversity and ecological factors by SPSS 21.0 software.RESULT:1、Collected 52 samples of different origin Hedysari folium,located in Gansu Hedysari Road estate area and infamous regions. According to the size of the area can be divided into three populations:1Wudu populations,Tanchang populations,Dingxi populations;2wild populations,cultivated populations;319 towns populations. 2、The results show that the best reaction system is a 25 μL volume of ISSR-PCR system with 10 x PCR Buffer(Mg2+)of 3.5 μL,DNA of 2 μL,primer of 2 μL,Tag DNA polymerase of 1.25 U,d NTPs of 2.0 μL, dd H2 O of 14.5 μL. The best number of cycles were 34.The 17 primers with polymorphism, a total of 17 primers amplified 484 bands, polymorphic bands(site) number were 479,the PPB was 98.97 %, the average value of amplifiedbands were 28.2 loci for each primers. 3、Three populations of Wudu, Tanchang, Dingxi of genetic diversity have differences in the different regions.The result of sort of three populations about genetic diversity level was:Tanchang>Wudu>Dingxi,showed that the population of Tanchang had wide genetic basis and better adaptability, the populations of Wudu, Tanchang, Dingxi had a rich genetic diversity. The populations of between wild Hedysari Radix and cultivated Hedysari Radix existed genetic variations. The result of sort of two populations about genetic diversity level was: wild Hedysari Radix > cultivated Hedysari Radix,showed that the population of wild Hedysari Radix had wide genetic basis and better adaptability, the populations of wild Hedysari Radix and ultivated Hedysari Radix had a rich genetic diversity. The populations of 19 towns of Hedysari Radix existed genetic variations. The populations of Anhua town had highest genetic diversity, the lowest Jiangtai Town The result showed that the population of Anhua had wide genetic basis and better adaptability, the populations of 19 towns had a rich genetic diversity. 4、Analysis on genetic differentiation of Hedysari Radix from different habits. The results showed that,the populations of Wudu, Tanchang, Dingxi overall genetic variation from 7.61 % variation existed between populations,genetic variation of 92.39 % existed within populations.The genetic variation of within populations account for a large proportion of gene flow coefficient was much greater than one, indicating that genetic differentiation of Hedysari Radix among populations obviously.The populations of genetic relationship between Wudu and Tanchang were closer, the populations of genetic relationship between Dingxi and Wudu and Tanchang were distant. The results showed that,the populations of wild Hedysari Radix and ultivated Hedysari Radix overall genetic variation from 5.44 % variation existed between populations,genetic variation of 94.56 % existed within populations.The genetic variation of within populations account for a large proportion of gene flow coefficient was much greater than one, indicating that genetic differentiation of Hedysari Radix among populations obviously. Wild populations and cultivated populations have differences on the genetic distance and genetic identity, as a wild populations,as a cultivated population. The results showed that,the populations of the populations of 19 towns overall genetic variation from 48.59% variation existed between populations,genetic variation of 51.41 % existed within populations.The genetic variation of within populations account for a large proportion of gene flow coefficient was much less than one, indicating that genetic differentiation of Hedysari Radix among populations obviously. The genetic distance between 19 towns populations of Hedysari Radix was 0.0692-0.4133, 0.6614-0.9332 range of genetic identity. The populations of genetic relationship between Anhua town and Guohe town were closer, the populations of genetic relationship between Zhangxian and Ganquan were farthest.The 19 towns populations have differences on the genetic distance and genetic identity. 19 populations clustered into two categories, the first category Zhangxian populations, other 18 populations of the second type, wherein the second category can be divided into four categories. 5、The genetic diversity of Hedysari Radix higher than Astragalus membranaceus(Fisch)Bge., Astragalus membranaceus(Fisch)Bge.var.mongholicus(Bge)Hsiao.,Pueraria lobata(Willd.)ohwi., Sophora alopecuroides L.,Mucuna pruriens var.Utilis., the genetic diversity of wild and cultivated Hedysari Radix higher than wild and cultivated Astragalus membranaceus, indicating that legume medicinal plant of Hedysari Radix with a wealth of genetic diversity Hedysari.6、The samples of wild and cultivated Hedysari Radix may be distinguished By DNA sample sites.The digital fingerprint code that was established by primer of 807,809,826,834, can identified 52 individual samples. 7、The result of correlations between genetic diversity and ecological factors showed that genetic diversity of Hedysari Radix exists obviously positive or negativecorrelations with the part of ecological factors. Longitude, extreme maximum temperature, annual average aridity, average frost-free period,≥0℃ accumulated temperature were the dominant ecological factors that influence genetic diversity of Hedysari Radix.CONCLUSION:The results showed that the stability of ISSR-PCR reaction system of Hedysari Radix is better and adapted to analysis genetic diversity of Hedysari Radix.The populations of Hedysari Radix from Gansu has a rich genetic diversity,the genetic differentiation of Hedysari Radix among populations was obviously.Through DNA loci could distinguish between wild and cultivation Hedysari Radix, DNA fingerprinting digital code of Hedysari Radix could identify 52 individual samples.The correlations between genetic diversity of Hedysari Radix and the part of ecological factors were closely.Longitude,extreme maximum temperature,the annual average,average frost-free period and ≥0 ℃ accumulated temperature as growth outside ecological factors on genetic diversity Hedysari Radix population had more significant selection pressure. |
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